Evo m mlv rt mix tracking kit

The GEPIA database (http://gepia.cancer-pku.cn/) consists of bulkRNA-seq samples derived from the TCGA, and GTEx database was used to verify the gene expression level of TPRGRS (36 (link)). P<0.05 was considered to be statistically significant. We applied Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) to validate the gene expression level of TPRGRS in tumor and normal cells. Two glioblastoma cell lines (U251 and U87) and one normal human astrocytes cell line (NHA) were purchased from Procell (Wuhan, China). Total RNA was extracted using AG RNAex Pro reagent (Accurate Biology) from U251, U87, and NHA. The Evo M-MLV RT Mix Tracking kit (Accurate Biology) was used for reverse transcriptase reaction, and SYBR-Green (Accurate Biology) was used for detection. The mRNA expression level of ARMC10, AUTS2, EN1, EREG, ERP29, HOXA2, HOXA5, HOXA7, HSPA5, LAP3, MDK, MTRF1L, NBEAL1, SLC6A6, and SLC37A3 was normalized by GAPDH. The primers of the fifteen genes were listed in Supplementary Table S1. Fold differences were calculated for each group using normalized CT values.

Total RNA from cells was extracted by Trizol (Invitrogen) following the manufacture's introduction. The concentration of RNA was measured with a Nanodrop‐2000 spectrophotometer (Thermo Scientific, USA). For analysis of mRNA expression, 1 µg total RNA was reverse transcribed using EvoM‐MLV RT Mix Tracking Kit (AG11734, Accurate Biotechnology, China), and qPCR was performed using SYBR Green Premix Pro Taq HS qPCR Tracking Kit (AG11733, Accurate Biotechnology, China). β‐actin was used as a housekeeping gene and relative expression of the target gene was determined using the 2 ^‐ΔΔCt method.